The Efficacy And Mechanism Of Surface-Anchored Nanogel Coating In Improving Stem Cells Survival And Infarcted Myocardium Repair

Purpose:Stem cell therapy has great potential in regenerative medicine.However,the low survival and engraftment rates of transplanted stem cells limit its effectiveness.Anchoring the nanogel on the surface of stem cells could improve the stress resistance in the hypoxic and ischemic environment of the infarcted heart.This study focused on the efficacy and mechanisms of stem cells coated with nanogel in the treatment of myocardial infarction.Methods and results:The polysialic acid system was used to anchor the microbial transglutaminase(m TG)to the cell membrane,which catalyzed the formation of nanogel on the surface of stem cells.The hydrogen nuclear magnetic resonance and the solid-state nuclear magnetic resonance characterized the successful cross-linking of the hydrogel.The scanning electron microscope of the hydrogel showed its porous structure.The nanogel coating did not affect the stem cell size,differentiation,migration,and the ability to take up external environmental substances,and basically had no effect on the paracrine function of stem cells.After transplanting stem cells into the rats suffering from the myocardial infarction,we found that the Gel-MSC(MSC coated with the nanogel)had less apoptosis than the MSC.Benefiting from it,more survived Gel-MSCs could better reduce cardiac cells apoptosis,promote angiogenesis and reduce infarct size compared to the MSC.The RNA sequencing and pathway analysis revealed that the nanogel coating mainly affected the signaling pathway of tumor necrosis factor α(TNFα)interaction with its receptor.The adhesion and binding experiments of MSC and red blood cells confirmed that the surface nanogel could weaken the interaction between TNFα and TNFR1.And the Gel-MSC enhanced the expression of Bcl-2/Bax and reduced the expression of pro-apoptotic gene Bak compared with the MSC under TNFαstimulation,indicating that nanogel coating inhibited apoptosis by reducing the interaction between TNFα and TNFR1.We also found that after TNFα stimulation,the nanogel could improve the length of mitochondria by transmission electron microscopy,enhance maximum oxygen consumption and intracellular ATP content compared to the MSC;tetramethylrhodamine methyl ester(TMRM)staining showed that Gel-MSC had higher mitochondrial membrane potential than the MSC after TNFα stimulation.These results indicated that the nanogel coating could improve the integrity and function of mitochondria under the condition of TNFα-induced cell damage.Then we found that the expression of optic atrophy 1(OPA1)and mitochondrial fusion protein 2(MFN2)in the MSC group after TNFα stimulation significantly reduced,and the activation of the upstream signaling pathways IκB/p50NFκB and PGC-1α significantly reduced while signaling pathways activation did not reduce obviously in the Gel-MSC group after TNFα stimulation,indicating that the nanogel improved the function and integrity of mitochondria under the condition of TNFα-induced cell injury through the PGC-1α/MFN2 pathway and the IκB/p50NFκB/OPA1 pathway,thereby improving the cell survival.Knocking down the expression of OPA1 or MFN2 could reverse the protective effect of nanogel coating on the cell survival;detecting the content of intracellular ROS and TMRM staining also showed that the protective effect of the nanogel on mitochondria disappeared,confirming that OPA1 and MFN2 were necessary for the nanogel coating to improve MSC survival and mitochondrial function after TNFα stimulation.Conclusion:In this study,we used the polysialic acid system to anchor microbial transglutaminase to the cell membrane,which catalyzed the formation of the nanogel coating on the surface of stem cells.The nanogel coating on the surface of stem cells did not affect the cell size,differentiation,migration,and the ability to take up external environmental substances,and basically had no effect on the paracrine function of stem cells.The nanogel could protect stem cells to reduce apoptosis caused by harsh environments so that more stem cells could exhbit the therapeutic role,thereby reducing cardiac cells apoptosis,promoting angiogenesis,reducing infarct size,and promoting post-infarction myocardium repair.The mechanism was that the nanogel coating reduced the binding of TNFα and TNFR1 and enhanced the IκB/p50NFκB and PGC-1αresponse pathways,thereby enhancing the expression of OPA1 and MFN2 to improve mitochondrial homeostasis,which contributed to the survival of stem cells in the area of myocardial infarction.